The present invention relates generally to materials and methods for use in immunological procedures for selection and differentiation of mammalian male and female cells. More specifically, the invention relates to a monoclonal anti-H-Y antibody, Hyclonalan, produced by a novel tumor cell line ATCC HB8116 and to uses of Hyclonalan in sex immunoselection procedures.
Sex immunoselection allows the separation and identification of male from female cells according to immunological reactions of antibodies specific for antigens on the cell surface. Such immunological reactions could mediate sex differentiation, for example, in a pool of embryos supplied by a female animal, or in a pool of X- and Y-bearing spermatozoa from a male animal. Gender-differentiated embryos or sperm may thereafter be utilized for embryo implantation or artificial insemination of the same or analogous species surrogate females for the preselection of the desired sex of the offspring.
As one example, in the cattle industry, methods for sex immunoselection could be of vital use in upgrading the nutritional characteristics and quantities of cattle raised to supply food and milk. While a cow of reproductive age will normally give birth to only one calf a year, which may be either a male or a female, accurate techniques of immunoselection could allow the birth of many genetically superior calves of a single sex as offspring of one genetically desirable cow. This could be accomplished by subjecting a group of hormonally induced ova from the genetically desirable cow either to fertilization and embryo immunoselection or to artificial insemination by sex-preselected sperm. Embryos thus obtained (e.g., all females) may be implanted into the uteri of surrogates and thereby the desirable genetic characteristics of the parent animals can be propagated with much greater frequency than is possible in nature. The ability to increase the reproductive capacity of genetically prized animals, especially dairy cattle, may be a key to solving the hunger problem which exists in many countries today due to insufficient number of meat-producers.
These technologies also show great promise in increasing the chances for survival of many endangered species. Animal experiments with interspecies embryo transfer have already shown that such techniques may ultimately prove successful in multiplying the flagging numbers of some endangered species of mammals by implanting sex-preselected, genetically desirable embryos from an endangered species into an analogous species surrogate.
In addition, sex immunoselection procedures are also directed at detection of microcellular sex determinants. In individuals with ambiguous gender development, such techniques are utilized to identify abnormal sex chromosome constitutions, particularly in human fibroblast cells.
Current investigation involves immunological techniques to detect the H-Y antigen, the cell surface component found on the male cells of all mammals. The locus of at least one of the genes responsible for H-Y expression is on the Y chromosome, and has been shown to be cross-reactive among numerous species ranging from fish to man. It has been proposed that the H-Y antigen may be the primary sex determinant and possibly the longsought inducer of testicular development in mammals. (Wachtel, et al., 1975; Wachtel and Koo, 1980); Koo, et al., "Application of Monoclonal Anti-H-Y Antibody for Human H-Y Typing," Human Genetics, 57: 64-67 (1981).
One of the limitations in current serological and immunological techniques used to detect the H-Y antigen on male cells, including those cells forming Y-bearing spermatozoa and male embryos is the source of antiserum. Antiserum is conventionally produced by injecting the antigen of interest into an immunologically responsive laboratory animal such as a mouse or rat and subsequently preparing antiserum from the blood of the animal, which will contain a mixture of antibodies developed against the antigen together with other antibody substances.
For example, Bryant, U.S. Pat. No. 4,191,749, granted Mar. 4, 1980, discloses a separation scheme for male- and female-determining spermatozoa utilizing a male-specific antibody. The antibody of this reference is prepared from the serum of female rabbits hyperimmunized with male rabbit epidermal cells. The antiserum, which must be complement inactivated, is "purified" by being repeatedly absorbed with washed female rabbit spleen cells and fractionated by agarose gel filtration to obtain the Immunoglobulin G (IgG) antiserum fraction.
Despite current scientific dispute concerning the presence of the H-Y antigen in haploid expression on the surface of spermatozoa, theoretically, X-bearing sperm will elute out of a column in which Bryant's Immunoglobulin G antiserum is coupled with solid phase immunosorbent material, while the Y-bearing sperm attach to the antiserum on the column. The Y-bearing sperm are then eluted out of the column separately with more antiserum solution in accordance with the principles of competitive binding.
Such conventionally-produced H-Y antiserum, although it contains H-Y antibody substances, is usually low-titered and contaminated with heteroantibody which will react with male and female cells of other species due to species-specific cell surface components which are not related to the H-Y antigen. Therefore clinical assays using the H-Y antiserum to detect the H-Y antigen on male cells or aid in separating male embryos and Y-bearing spermatozoa from female embryos and X-bearing spermatozoa will often yield ambiguous and inaccurate results in embryo transferral and fertilization experiments.
This ambiguity in anti-H-Y antiserum is also disadvantageous when used in serologic procedures for the detection of H-Y antigen expression in patients (both animal and human) that demonstrate ambiguous sexual development (hermaphroditic differentiation) or other physical conditions caused by genetic aberrancies in the sex chromosomes. Hermaphroditic differentiation is believed to be caused by the presence of Y-chromosome material as an intact Y chromosome in a mosaic cell line, as a minute part of extra material attached to an X chromosome or as an autosome. Because the chromosome segments and genes which form them are so small, the usual cytological assays cannot always detect them. Serological detection of H-Y antigen expression of these chromosome segments and genes is considered the best evidence of these genotypic abnormalities. However, the serological assays are only as precise in locating the genetic abnormality as is the anti-H-Y antiserum available for use. Wachtel, et al., "Serologic Detection of a Y-Linked Gene in XX Males and XX True Hermaphrodites," New England J. Med., 295: 750-754 (1976).
Researchers in this field have therefore turned to hybridoma techniques to produce tumor cell lines which will manufacture highly specific monoclonal H-Y antibody. Techniques used for the production of monoclonal antibody are well known in the art, and can be found described in Oi, V. T. and L. A. Herzenberg, "Immunoglobulin Producing Hybrid," Mishell, B. B. and S. M. Shiigi (eds.), Selected Methods in Cellular Immunology. San Francisco: W. H. Freeman Publishing, 1979. Lymphocytes removed from the spleen of an animal previously injected with the antigen of interest are allowed to fuse with myeloma cells in the presence of polyethylene glycol or a similar fusogen. Thousands of "hybrid" myeloma cells are produced from the fusion. The supernatant from growth of each "hybridoma" cell culture is tested for the presence of the desired antibody activity. When such activity is found in the supernatant of one cell culture, it is cloned by limiting dilutions, and the clones produced are individually assayed for supernatant activity.
To date, monoclonal antibody methodologies have produced monoclonal anti-H-Y antibodies of widely varying specificities and physiological characteristics. An H-Y antibody less than highly specific, however, will not provide an accurate detection of solely the H-Y antigen cell surface which will enable identification and separation of male from female cells and accurate prediction of the results of sex immunoselection techniques, or locate fragments of Y chromosomes in cells of patients with abnormal sex chromosome constitutions.
There exists, therefore, a substantial need for a highly specific monoclonal H-Y antibody to improve the accuracy of sex immunoselection of cells for purposes of artificial insemination and embryo transferral (particularly in the areas of upgrading animal herds and increasing the populations of endangered mammalian species) and for identification of genetic abnormalities.
Specifically incorporated by reference herein for the purposes of indicating the background of the invention and illustrating the state of the art are the following publications:
1. Goldberg, et al., "Serological Demonstration of H-Y (Male) Antigen on Mouse Sperm," Nature, 232: 478 (1971);
2. Kohler, G. and Milstein, C., "Derivation of Specific Anti-Body Producing Tissue Culture and Tumor Cells by Cell Fusion," Nature, 256: 495 (1975);
3. Koo, et al., "Application of Monoclonal Anti-H-Y Antibody for Human H-Y Typing," Human Genetics, 57: 64-67 (1981);
4. Krco, C. J., and Goldberg, E. H., "H-Y (Male) Antigen. Detection on Eight-Cell Mouse Embryos," Science, 193: 1134-1135 (1976);
5. Oi, V. T. and L. A. Herzenberg, pp. 351-372 in "Immunoglobulin Producing Hybrid," Mishell, B. B. and S. M. Shiigi (eds.), Selected Methods in Cellular Immunology. San Francisco: W. H. Freeman Publishing, 1979; and
6. Wachtel, et al., "Serologic Detection of a Y-Linked Gene in XX Males and XX True Hermaphrodites," New England J. Med., 295: 750-754 (1976).